The RING/U-box E3 protein BIR1 mediates ubiquitination of the barley leaf-growth repressor BROAD LEAF1.

Establishment of final leaf size in plants relies on the precise regulation of two interconnected processes, cell division and cell expansion. The barley protein BROAD LEAF1 (BLF1) limits cell proliferation and leaf growth in the width direction. However, how the levels of this potent repressor of leaf growth are controlled remains unclear. Here we used a yeast two-hybrid screen to identify the novel BLF1-interacting RING/U-box 1 (BIR1) E3 ubiquitin ligase that interacts with BLF1 and confirmed the interaction of the two proteins in planta. Inhibiting the proteasome caused overaccumulation of a BLF1-eGFP fusion protein when co-expressed with BIR1, and an in vivo ubiquitination assay in bacteria confirmed that BIR1 can mediate ubiquitination of BLF1 protein. Consistent with regulation of endogenous BLF1 in barley by proteasomal degradation, inhibition of the proteasome in BLF1-vYFP expressing barley plants caused an accumulation of the BLF1 protein. The BIR1 protein co-localized with BLF1 in nuclei and appeared to reduce BLF1 protein levels. Analysis of bir1-1 knock-out mutants suggested the involvement of the BIR1 gene in leaf growth control, although mainly on leaf length. Together, our results suggest that proteasomal degradation, in part mediated by BIR1, helps fine-tune BLF1 protein levels in barley.